Cas13b-mediated RNA targeted therapy alleviates genetic dilated cardiomyopathy in mice

Background Recent advances in gene editing technology have opened up new avenues for in vivo gene therapy, which holds great promise as a potential treatment method for dilated cardiomyopathy (DCM). The CRISPR-Cas13 system has been shown to be an effective tool for knocking down RNA expression in mammalian cells. PspCas13b, a type VI-B effector that can be packed into adeno-associated viruses and improve RNA knockdown efficiency, is a potential treatment for diseases characterized by abnormal gene expression. Results Using PspCas13b, we were able to efficiently and specifically knockdown the mutant transcripts in the AC16 cell line carrying the heterozygous human TNNT2R141W (hTNNT2R141W) mutation. We used adeno-associated virus vector serotype 9 to deliver PspCas13b with specific single guide RNA into the hTNNT2R141W transgenic DCM mouse model, effectively knocking down hTNNT2R141W transcript expression. PspCas13b-mediated knockdown significantly increased myofilament sensitivity to Ca2+, improved cardiac function, and reduced myocardial fibrosis in hTNNT2R141W DCM mice. Conclusions These findings suggest that targeting genes through Cas13b is a promising approach for in vivo gene therapy for genetic diseases caused by aberrant gene expression. Our study provides further evidence of Cas13b’s application in genetic disease therapy and paves the way for future applicability of genetic therapies for cardiomyopathy.


Background
RNA-based therapeutics have been investigated in the past decade for the treatment of various diseases, such as cardiovascular disorders [1,2], cancer [3][4][5], central nervous system disease [6][7][8] and endocrine system disease [9][10][11].Conventional methods targeting transcripts using RNA interference (RNAi) or antisense oligonucleotides have significant nonspecific and off-target effects [12,13].However, a member of a family of RNA-targeting CRISPR effectors called Cas13 has demonstrated a robust ability to downregulate cellular RNAs in mammalian cells in vitro [14][15][16][17].Cas13b represents a significant improvement over existing RNA-targeting methods due to its lower off-target effects, with the PspCas13b subtype displaying the most potent RNA knockdown efficiency [18].Furthermore, PspCas13b is a type VI-B effector with a small size that can be packaged into adeno-associated viruses (AAVs), making it a promising therapeutic tool for diseases characterized by aberrant gene expression.
Dilated cardiomyopathy (DCM) is a cardiac disorder characterized by dilatation and impaired contraction of the left ventricle or both ventricles and is a leading cause of heart failure and heart transplantation [19][20][21].Familial DCM accounts for 20-48% of cases, a large proportion of which are principally results of mutations in the genes encoding sarcomeric proteins in cardiomyocytes [22,23].Troponin T, encoded by the TNNT2 gene, is one such sarcomeric protein.Troponin T binds troponin C and troponin I to form the troponin complex that regulates muscle contraction via calcium sensitivity through conformational changes in the interaction between cardiac actin and myosin heavy chain [24,25].The TNNT2 R141W variant was first discovered in a five-generation DCM family using the combination of Sanger sequencing of candidate genes and restriction-endonuclease analysis [24].To investigate the functional consequences of this variant, Zhang et al. [26] generated a humanized TNNT2 R141W transgenic mouse model (R141W mice), which exhibits a progressive and severe form of DCM by the age of 4 months.Numerous studies have delved into the pathological mechanisms pertaining to the human TNNT2 R141W (hTNNT2 R141W ) mutation in R141W DCM mice [27][28][29][30].Nevertheless, gene therapy research targeting DCM caused by the TNNT2 mutation needs further study.
Heterozygous mice with one disrupted copy of the Tnnt2 gene exhibit a mild decrease in transcript expression but no discernable changes in protein expression or phenotype [31].In addition, current evidence in the Clin-Var and DECIPHER databases does not report TNNT2 haploinsufficiency as a cause of cardiomyopathy.Therefore, we propose that RNA-targeted therapy targeting mutant TNNT2 mRNA may be a promising approach for treating DCM caused by TNNT2 mutations.
In this study, we established the PspCas13b system to target hTNNT2 R141W mRNA in a R141W DCM mouse model.We found that adeno-associated virus vector serotype 9 (AAV9)-delivered PspCas13b and single guide RNA (sgRNA) disrupted hTNNT2 R141W mRNA expression and alleviated the cardiac dysfunction in hTNNT2 R141W transgenic mice.These findings are encouraging and will pave the way for future genetic cardiomyopathy therapies.

A hTNNT2 R141W transgenic mouse model with DCM
To determine the mechanistic role of the hTNNT2 R141W variant in DCM, Zhang and colleagues [26] generated a transgenic mouse model expressing the human TNNT2 R141W protein, which was maintained on a C57BL/6J genetic background (Fig. 1A).Through genotyping, we amplified a fragment consistent with the positive control in R141W mice (Fig. 1B).By performing HpaII enzyme digestion on the hTNNT2 R141W cDNA amplification product, fragments of 147 bp and 66 bp + 44 bp sizes were cleaved from the R141W mouse cDNA amplicon (Fig. 1C).The expression of hTNNT2 R141W was specifically controlled by the α-MHC promoter in cardiomyocytes.The mRNA levels of TNNT2 in the heart, liver, spleen, lung, kidney, skeletal muscle, and brain were confirmed by quantitative realtime PCR (qRT-PCR), suggesting that the hTNNT2 R141W gene was specifically expressed in the heart (Fig. 1D).At four months of age, the transgenic mice exhibited ventricular chamber enlargement and systolic dysfunction (Fig. 1E, F).Here we examined the DCM-associated phenotype of the R141W mouse model, implying its potential use for subsequent research.

Specific knockdown of hTNNT2 R141W mRNA in vitro using PspCas13b
In order to effectively knockdown hTNNT2 R141W transcripts for the treatment of DCM, we designed to use AAV9 to deliver PspCas13b along with specific sgRNA into hTNNT2 R141W transgenic mice (Fig. 2A).First, to optimize the PspCas13b-mediated knockdown efficiency of the mutant TNNT2 R141W transcript, we screened for the most effective sgRNAs targeting the TNNT2 mutant site.Ten TNNT2-sgRNAs and PspCas13b vectors were cotransfected into TNNT2 R141W 293T cells that had been infected with EF1α-TNNT2 R141W -puro lentivirus (Fig. 2B).Forty-eight hours after transfection, qRT-PCR assays revealed that cotransfection of TNNT2-sgRNA2 (sgTNNT2) and the PspCas13b vector decreased TNNT2 R141W expression by 75.9 ± 0.97% (Fig. 2C).
To further investigate the specificity of PspCas13bmediated knockdown of TNNT2 R141W , we established an AC16 cell line carrying the heterozygous TNNT2 R141W mutation (Fig. 2D).Cells were infected with PspCas13b and sgTNNT2 or sgCtrl as a control.Two days after infection, qRT-PCR analysis showed that total TNNT2 expression was reduced to 72.4 ± 3.1% of that in control cells (Fig. 2E, G).We also ligated the RT-PCR product of the TNNT2 R141W fragment into a T vector and selected 100 colonies for Sanger sequencing.Analysis of the sequencing results revealed that in the sgTNNT2 group, 79 colonies had a WT sequence, while 21 colonies had a mutant  sequence.In contrast, in the control group, 53 colonies had a WT sequence, and 47 colonies had a mutanttype sequence (Fig. 2H).These results suggest that the CRISPR-Cas13b system can efficiently and specifically knock down TNNT2 R141W transcript expression in vitro.
To detect the potential off-target effects, sgTNNT2s and PspCas13b vectors were cotransfected into TNNT2 R141W 293T cells that had been infected with EF1α-TNNT2 R141W -puro lentivirus.Forty-eight hours after transfection, cells were collected for RNA extraction and subsequent transcriptome-wide mRNA sequencing to detect significant differentially expressed genes.Transcriptome analysis showed that TNNT2 was specifically downregulated while the transcriptional level of other genes remained unchanged 2 days after transfection (Fig. 2I).

PspCas13b effectively knocked downhTNNT2 R141W transcript expression in vivo
To investigate the feasibility of PspCas13b-mediated knockdown of hTNNT2 R141W mRNA expression in vivo, we injected AAV9s encoding PspCas13b and sgTNNT2 (AAV9-PspCas13b and AAV9-sgTNNT2) into 4-month-old R141W mice via tail vein injection (Fig. 3A).As shown in the schematic diagram, mice received twice tail vein injections, with the second injection administered 14 days after the initial injection.Samples were obtained on days 1, 4, 7, 15, 21, and 28 after the first AAV9 injection to detect hTNNT2 R141W transcript expression levels.On the first day after AAV9-PspCas13b injection, hTNNT2 R141W mRNA levels in cardiomyocytes were 54.4 ± 4.7% of those in cardiomyocytes without AAV9 injection.As PspCas13b vector expression in cardiomyocytes gradually declined, hTNNT2 R141W mRNA levels gradually returned to normal levels by 7 days after injection.The second AAV9-PspCas13b injection achieved similar efficiency of hTNNT2 R141W knockdown as observed in the first injection (Fig. 3B).Furthermore, qRT-PCR analysis revealed significant upregulation of mouse Tnnt2 (mTnnt2) expression on the fourth day after the first injection and at several subsequent time points (Fig. 3C).These results demonstrate that PspCas13b can effectively knock down the expression of the mutant hTNNT2 R141W transcript in vivo.As hTNNT2 R141W expression decreased, mTnnt2 expression increased to compensate.

PspCas13b-mediatedhTNNT2 R141W knockdown regulates intracellular Ca 2+ and sarcomere dynamics in cardiomyocytes
Reportedly, the p.R141W mutation in hTNNT2 causes DCM by decreasing Ca 2+ sensitivity through increased affinity for tropomyosin [35].To assess the functional improvement mediated by hTNNT2 R141W knockdown on cardiomyocyte contractile function, we measured Ca 2+ dynamics and contractile properties in PspCas13bmediated hTNNT2 R141W knockdown cardiomyocytes and compared them with those in WT and untreated R141W mouse cardiomyocytes.The results showed that the size of R141W mouse cardiomyocytes significantly decreased after injection of the AAV9-PspCas13b system (Fig. 4A).Additionally, the sarcomere length of cardiomyocytes in WT and AAV9-PspCas13b-injected R141W mice was significantly shorter than that in R141W mice (Fig. 4B).

PspCas13b-mediatedhTNNT2 R141W knockdown restores cardiac function
To evaluate the therapeutic effects of PspCas13b-mediated knockdown of hTNNT2 R141W in R141W DCM mice, we administered AAV9 vectors containing PspCas13b and sgTNNT2 through tail vein injection to 4-monthold R141W mice (Fig. 3A).Histological characteristics and cardiac function were assessed in three groups (WT, R141W, and R141W + PspCas13b) four weeks after injection.The hTNNT2 R141W mutation in cardiomyocytes can result in an increased heart-to-body-weight (HW/BW) ratio and increased cardiomyocyte area, both of which are indicators of DCM.After treatment with PspCas13b, we observed a reduction in heart weight in R141W mice (Fig. 5A, B).Wheat germ agglutinin (WGA) staining showed a decrease in cardiomyocyte area in PspCas13btreated R141W mice (Fig. 5C, D).Echocardiography  revealed that R141W mice significantly reduced ejection fraction (EF), fractional shortening (FS), and systolic left ventricular posterior wall thickness (LVPW;s), while the systolic left ventricular internal diameter (LVID;s) was significantly increased compared to that of the WT group (Fig. 5E-I).Following knockdown of hTNNT2 R141W mRNA expression with PspCas13b for four weeks, EF, FS and LVID;s were significantly restored (Fig. 5F-H).
Masson staining revealed that cardiac interstitial collagen content increased in R141W mice, while PspCas13b treatment significantly reduced interstitial collagen deposition in R141W mouse myocardium (Fig. 5J, K). qRT-PCR demonstrated decreased mRNA levels of collagen type I (Col1a1) and periostin (Postn), the mRNA levels of fibrosis markers, in the myocardium of R141W + Psp-Cas13b mice compared with R141W mice (Fig. 5L, M), indicating the efficacy of PspCas13b treatment in alleviating cardiac interstitial fibrosis in R141W mice.In addition, the mRNA levels of heart failure markers, natriuretic peptide type A (Nppa) and natriuretic peptide type B (Nppb), which were increased in R141W mice, were sharply decreased after the PspCas13b treatment (Fig. 5N, O).Our findings suggest that CRISPR-Cas13b-mediated knockdown of hTNNT2 R141W mRNA expression can restore cardiac function, reduce cardiac interstitial fibrosis, and ameliorate heart failure in DCM mice.

Discussion
Cas13b is a CRISPR-associated enzyme known for its high cleavage activity and targeting specificity, which make it an excellent candidate for therapeutic applications.In this study, based on the R141W DCM mouse model, we used the CRISPR-Cas13b system to knock down the mRNA of hTNNT2 R141W .Our results demonstrated that this approach restored cardiac function in R141W DCM mice, highlighting the potential of Cas13b as a promising tool for treating this debilitating condition.
In recent years, CRISPR systems have been explored for the treatment of genetic diseases and DNA virus replication due to their efficient DNA editing capabilities [36][37][38][39].However, the potential risks associated with permanent DNA alterations resulting from the standard CRISPR-Cas9 editing system have limited its clinical application [14-16, 18, 40].In vivo gene therapy holds great promise for the treatment of cardiovascular diseases, including DCM [41].RNAi-based approaches, such as siRNA and shRNA treatment, have shown efficacy in improving cardiac function and reducing myocardial fibrosis in animal models of DCM [1,13,42].However, the off-target effects associated with RNAi-based therapies limit their clinical application.In contrast, RNA-targeting CRISPR systems, such as the CRISPR-Cas13b system, offer a promising alternative with potentially lower risks for clinical use.CRISPR-Cas13b system, which requires single guide RNA to target the intended sequence, was first reported by Feng Zhang in 2017 [18].CRISPR-Cas13b system has demonstrated a consistent knockdown efficiency of > 80% for the expression of various endogenous mRNAs and lncRNAs [15,18], with markedly higher specificity than that of spacer-matching shRNAs, and no detectable off-target effects in comparison to the hundreds observed for RNAi.These characteristics position Cas13b as a potentially transformative therapeutic tool for the treatment of diseases marked by abnormal gene expression patterns.The CRISPR-Cas13b system is modifiable to enhance viral RNA clearance, suppress viral replication, and circumvent its mutational escape in order to achieve the goal of controlling viral infections [43][44][45][46].In the treatment of cancer, it is possible to engineer the CRISPR-Casl3b system to regulate cellular methylation, which in turn inhibits tumor progression and controls tumor metastasis [47,48].Rashnonejad et al. proposed that DUX4 silencing is the most direct route to Facioscapulohumeral muscular dystrophy (FSHD) therapy and they developed an AAV6-CRISPR-Cas13b strategy to silence DUX4 mRNA.Intramuscular delivery of an AAV6 vector encoding a PspCas13b enzyme and DUX4-targeting guide RNAs reduced DUX4 mRNA by > 50% and improved histopathological outcomes in FSHD mice [49].As of now, there are no reported applications of the CRISPR-Cas13b system in the treatment of cardiovascular diseases.Our study utilized AAV9 delivery to demonstrate that the CRISPR-Cas13b system can effectively cleave targeted mRNA in vivo, knocking down the hTNNT2 R141W mRNA expression and subsequently alleviating genetic DCM in mice.This marks a groundbreaking advancement, as this is the first report of the application of the Cas13b system in treating cardiomyopathy or even genetic cardiovascular disorders to our knowledge, highlighting the potential of Cas13b-mediated gene therapy for cardiomyopathies and potentially genetic diseases that require downregulation of aberrant gene products.

Conclusions
Overall, our findings indicate that AAV9-mediated administration of PspCas13b can effectively suppress hTNNT2 R141W mRNA expression and improve cardiac function in a murine model of DCM.These results support the potential application of the RNA-targeting CRISPR-Cas13b system as a promising therapeutic strategy for this disorder.

Animals
The α-MHC-hTNNT2 R141W DCM transgenic mice (referred to as R141W mice) were generated previously in the Laboratory of Animal Science of Peking Union Medical College [26].All of the mice used in this study were maintained on a C57BL/6J genetic background, and all animal experimental procedures were approved by the Ethics Committee for Animal Research of Fuwai Hospital and adhered to the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85 − 23, revised 1996).Mice were genotyped by PCR with the forward primer 5'-GAACAGGAGGAAGGCTGAGGATGAG-3' and the reverse primer 5'-TATTTCCAGCGCCCGGT-GACTTTAG-3' .The hTNNT2 R141W cDNA was amplified for restriction endonuclease analysis with the forward primer 5'-TTCATGCCCAACTTGGTGC-3' and the reverse primer 5'-CTCTCTTCAGCCAGGCGGTTC-3' .Restriction endonuclease HpaII was obtained from New England Biolabs, and the digestion was conducted at 37 ℃ for 3 h and separated by electrophoresis.The hTNNT2 R141W cDNA amplification product can be cut into 3 fragments of 147 bp and 66 bp + 44 bp sizes, while the wild-type cDNA cannot be digested and remains unchanged as a product of 260 bp.

Cell line
The human embryonic kidney 293 (HEK-293T) cell line and human AC16 cardiomyocytes were obtained from the China Infrastructure of Cell Line Resource (NICR) and cultured in DMEM with 10% FBS and 1% penicillin/ streptomycin in a 37 °C incubator under 5% CO 2 .

In vitro sgRNA testing
In accordance with Zhang et al. 's study [15], we have designed 10 sgRNAs specifically targeting the mutation site.The sequences of these sgRNAs are listed in Table 1.To facilitate their binding, we have added a CACC sticky end to the 5' end of the designed sgRNA sequences and a CAAC sticky end to the 5' end of the reverse complementary sequences of the sgRNAs.Subsequently, we synthesized 10 pairs of sgRNA oligos, which were further diluted to 100 pM.In the reaction system utilizing T4 PNK (NEB # M0201V), one pair of sgRNA oligos underwent a gradient annealing process from 95 °C to 25 °C, resulting in the formation of dsDNA with double sticky ends.The obtained product was then diluted 1:250 for subsequent applications.
The sgRNA vector (pC0043-PspCas13b crRNA backbone, Addgene #103,854) was enzymatically digested using the BbsI restriction enzyme, and the resulting digested products were recovered utilizing the TIANgel purification kit (TIANGEN #DP219).Following the recovery, the digested sgRNA vector was ligated with the annealed sgRNA using T4 ligase, which proceeded overnight at 16 °C.For the subsequent transfection step, 2400 ng of constructed sgRNA plasmid and 1200 ng of PspCas13b plasmid (pC0046-EF1a-PspCas13b-NES-HIV, Addgene #103,862) were cotransfected into TNNT2 R141W 293T cells in a 12-well plate.These cells had previously undergone infection with the lenti-TNNT2 R141W -Puro lentivirus and were selected using puromycin.Following the 48-hour transfection period, total RNA was extracted using the Trizol method, and cDNA was synthesized for subsequent qRT-PCR analysis to assess knockdown efficiency.

Testing of the specificity of the PspCas13b system
We developed two mini-constructs containing a fluorescent reporter gene linked to either WT-TNNT2 or MT-TNNT2 R141W , as shown in Fig. 2E.These constructs were designed based on the human TNNT2 mRNA sequence and differed in the position of the mutation recognition site.To create heterozygous conditions, we cotransfected the two reporter alleles and PspCas13b-sgRNA2 duplexes into cultured HEK-293 cells.We then conducted qRT-PCR to detect the expression levels of GFP and RFP, which enabled us to assess the effectiveness of the CRISPR-Cas13b system in targeting and suppressing the mutant allele while preserving expression of the WT allele.
The WT AC16 cell line was converted into a mutant cell line with a heterozygous TNNT2 R141W allele using SPRY-CBE4max (Fig. 2D).PspCas13b and sgRNA2 were cotransfected into this cell line, and total TNNT2 expression levels were measured using qRT-PCR.PCR amplicons containing the R141W mutation site were then cloned into the pBackZero-T Vector (TAKARA, Beijing, China) for sequencing via Sanger sequencing, and the results were analyzed using Chromas.

Testing of the off-target effects of the PspCas13b system
We cotransfected sgTNNT2s and PspCas13b vectors into TNNT2 R141W 293T cells that had been infected with EF1α-TNNT2 R141W -puro lentivirus.48 h after transfection, cells were collected for RNA extraction and subsequent transcriptome-wide RNA-Seq.RNA-seq data was analyzed as previously described [50] and presented as the mean of all repeats.The mRNA sequencing (highthroughput) was performed using the Illumina Genome Analyzer and the adapters were removed using Trimmomatic (v0.36) during sequencing.The Hisat2 (v2.0.0) was used to map qualified reads to the mouse reference genome (mm10) with default parameters.Then, the expression levels of all mapped genes were estimated by Stringtie (v2.0) and the gene expression abundances were indicated by FPKM (fragments per kilobase of transcript per million fragments mapped).

AAV9 production and tail vein injection
AAV9-EF1α-PspCas13b and AAV9-U6-sgTNNT2 were produced by transfecting HEK293T cells with polyethylenimine (PEI) at a concentration of 50 µg/mL.Briefly, plasmids encoding the viral vector, packaging proteins, and helper reagents were cotransfected into HEK293T cells using PEI.The viruses were then harvested from the cell culture medium 3-7 days after transfection and purified by ultracentrifugation through a cesium chloride gradient.The virus-containing fractions were collected, dialyzed against phosphate-buffered saline (PBS), and concentrated using Amicon Ultra centrifugal filter units (100 kDa cutoff ).
To evaluate the therapeutic efficacy of these viruses in vivo, we performed tail vein injections of 4 × 10^12 AAV9 particles (determined by qRT-PCR) into 4-month-old mice, which enabled efficient systemic delivery to the heart.These optimized procedures have been extensively validated and provide robust and reproducible results for studies utilizing RNA-targeting CRISPR-Cas13b technology.

RNA extraction and quantitative real-time PCR
Total RNA was extracted from HEK-293T cells or heart tissues using TRIzol reagent (Invitrogen, CA, USA) according to the manufacturer's instructions.The RNA samples were then reverse-transcribed into cDNA using PrimeScript RT Master Mix (Takara, RR036A).qRT-PCR analysis was performed using PowerUp™ SYBR Green Mastermix (Applied Biosystems, Shanghai, China) on a StepOnePlus Real-Time PCR System (Applied Biosystems).The qRT-PCR cycling conditions consisted of an initial denaturation step at 95 °C for 10 min followed by 40 cycles of denaturation at 95 °C for 15 s and annealing/ extension at 60 °C for 1 min.Relative gene expression levels were calculated using the 2-∆∆Ct method, with β-actin used as the internal control for normalization.The primers used in this research are listed in Table 2.

Histology
Mouse hearts were harvested and fixed in 4% paraformaldehyde at room temperature for 24 h.Subsequently, the hearts were dehydrated through an ethanol and xylene series and finally paraffin-embedded.The paraffin-embedded hearts were sliced continuously into 5-mm-thick sections, with five sections collected from each heart.Standard procedures were used to perform hematoxylin and eosin (H&E) and Masson's trichrome staining.

Immunofluorescence assay
Immunofluorescence staining was performed on paraffin-embedded tissue sections to visualize the localization and distribution of specific proteins.Deparaffinization, antigen retrieval, and blocking of nonspecific background staining were performed as previously described [51][52][53][54].The tissue sections were incubated with primary antibodies overnight at 4 °C.After washing three times with PBS, the slides were incubated with a fluorescently labeled secondary antibody appropriate for the primary antibody for 1 h at room temperature.The slides were then washed again three times in PBS, counterstained with 4' ,6-diamidino-2-phenylindole (DAPI, Sigma), and mounted with VECTASHIELD mounting medium (Vector Labs, CA, USA).The primary antibody used in this study was anti-WGA conjugated with Alexa Fluor 647 (Invitrogen W32466, 20 mg/mL).The secondary antibody used was donkey anti-rabbit conjugated with Alexa Fluor 488 (Invitrogen A-21,206, 1:500 dilution).Fluorescence images were obtained using a ZEISS LSM800 confocal laser scanning microscope, and acquisition settings were kept consistent between samples.All analyses were performed while blinded to the treatment groups, and representative images were chosen to demonstrate key findings.

Assessment of myofilament sensitivity to Ca 2+
Single ventricular myocytes were isolated using a modified version of a previously described technique [55].Animals were euthanized and their hearts were rapidly removed and mounted in Langendorff mode.The hearts were perfused with HEPES-based salt solution (isolation The perfusate contained 0.75 mmol/L Ca 2+ .After stabilization, perfusion was continued for 4 min with Ca 2+ -free isolation solution containing 0.1 mM EGTA followed by 6 min with isolation solution containing 0.05 mmol/L Ca 2+ , 0.75 mg/mL collagenase (type 1; Worthington Biochemical Corp., USA), and 0.075 mg/mL protease (type X1V; Sigma, Germany).The ventricles were then excised from the heart, minced, and gently shaken in collagenase-containing isolation solution supplemented with 1% bovine serum albumin (BSA).Cells were filtered from the solution at 4-minute intervals and resuspended in isolation solution containing 0.75 mmol/L Ca 2+ .Ventricular myocytes were isolated from five control and five mutant mice.

Echocardiography
Left ventricular systolic function was analyzed by echocardiography one month after AAV9 injection using a digital ultrasound system (Vevo2100 Imaging System, Visual Sonics, Toronto, Canada).The conventional left ventricle functional parameter measurements included EF, FS, end-diastolic diameter, and end-systolic diameter.

Cell surface area measurement
The cell surface area of WGA-stained cells was measured by ImageJ.Approximately 100-200 cardiomyocytes in 20-50 fields of each heart tissue were examined in each experiment.

Statistical analysis
The data are presented as the mean ± standard error of the mean (SEM).Student's t test was used to analyze the results of the control and experimental samples.The results with a p value ＜ 0.05 were considered statistically significant.

Fig. 2
Fig. 2 Specific knockdown of hTNNT2 R141W in vitro using PspCas13b.(A) Schematic showing the experimental procedure for the delivery of PspCas13b and sgTNNT2-expressing AAV9s into hTNNT2 R141W transgenic mice.(B) Schematic representation of the mechanism of the PspCas13b-sgRNA system in silencing the expression of the mutant TNNT2 R141W transcript.(C) qRT-PCR demonstrating successful knockdown of hTNNT2 R141W in TNNT2 R141W -293T cells (n = 4 per group).***p < 0.001 according to Student's t test.(D) Schematic diagram of the process for constructing an TNNT2 WT/R141W -AC16 cell line using SpRY-CBEmax.(E) Schematic representation of the process of PspCas13b-specific knockdown of mutant transcript expression in the TNNT2 WT/R141W -AC16 cell line and the HEK-293T cell line with reporter alleles.(F) Assessment of knockdown of WT (green) and mutant (red) mRNA expression in HEK-293T cells transfected with reporter alleles and PspCas13b-sgRNA2 normalized to the levels observed in cells transfected only with the reporter alleles (n = 3 independent experiments).(G) qRT-PCR quantification of total TNNT2 mRNA in TNNT2 WT/R141W -AC16 cells (n = 6 independent experiments).*p < 0.05 according to Student's t test.(H) Sanger sequencing of T vectors with WT and hTNNT2 R141W transcripts.(I) Expression levels in log2 (fragments per kilo base per million mapped reads [FPKM] + 1) values of all detected genes in RNA sequencing (RNA-seq) libraries of PspCas13b-TNNT2 (y axis) compared to PspCas13b control (x axis) (n = 3 independent replicates for both groups)

Fig. 3
Fig. 3 Effective knockdown of hTNNT2 R141W mRNA expression through PspCas13b in vivo.(A) Schematic of the experimental procedure.(B) Quantification of hTNNT2 mRNA levels in hearts from AAV9-PspCas13b-sgTNNT2-injected (n = 6) and noninjected R141W mice (n = 3).(C) Quantification of the relative expression of the mouse Tnnt2 gene in DCM and DCM + AAV9 cardiomyocytes by qRT-PCR using the 2 -∆∆Ct method (n = 3).The bar graphs show the mean ± SEM.NS is not significant; *p < 0.05 according to Student's t test

Table 1
The sequences of sgRNAs designed for the targeted mutation site

Table 2
Primers used in this research for qRT-PCR ) at a physiological temperature (36-37 °C) and a constant flow rate adjusted according to heart weight. solution